Abstract: A gene editing system comprising: (a) a Type V CRISPR nuclease polypeptide or a first nucleic acid encoding the Type V CRISPR nuclease polypeptide; (b) a reverse transcriptase (RT) polypeptide or a second nucleic acid encoding the RT polypeptide; (c) a guide RNA (gRNA) or a third nucleic acid encoding the gRNA, wherein the gRNA comprises one or more binding sites recognizable by the Type V CRISPR nuclease (CRISPR nuclease binding sites) and a spacer sequence specific to a target sequence within a genomic site of interest, the target sequence being adjacent to a protospacer adjacent motif (PAM); and (d) a reverse transcription donor RNA (RT donor RNA) or a fourth nucleic acid encoding the RT donor RNA, wherein the RT donor RNA comprises a primer binding site (PBS) and a template sequence.

Abstract: A Streptococcus canis Cas9 (ScCas9) ortholog and its engineered variants, possessing novel PAM specificity, is an addition to the family of CRISPR-Cas9 systems. ScCas9 endonuclease is used in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence. A novel DNA-interacting loop domain within ScCas9, and other Cas9 orthologs, such as those from Streptococcus gordonii and Streptococcus angionosis facilitates a divergent PAM sequence from the “NGG” PAM of SpCas9.

Abstract: A system for genetic editing of a transthyretin (TTR) gene, comprising (i) a Cas12i2 polypeptide or a first nucleic acid encoding the Cas12i2 polypeptide, and (ii) an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an TTR gene. Also provided herein are methods for editing a TTR gene using the gene editing system disclosed herein and/or for treating diseases associated with the TTR gene.

Abstract: Engineered Streptococcus canis Cas9 (ScCas9) variants include an ScCas9 protein with its PID being the PID amino acid composition of Streptococcus pyogenes Cas9 (SpCas9)-NG, an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 in its amino acid sequence (Sc+), and an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 and a substitution of residues ADKKLRKRSGKLATE [SEQ ID No. 4] in position 365-379 in the ScCas9 open reading frame (Sc++). Also included are CRISPR-associated DNA endonucleases with a PAM specificity of 5?-NG-3? or 5?-NNG-3? and a method of altering expression of a gene product by utilizing the engineered ScCas9 variants.

Abstract: Applications of a Streptococcus Cas9 ortholog from Streptococcus macacae (Smac Cas9), possessing minimal adenine-rich PAM specificity, include an isolated Streptococcus macacae Cas9 protein or transgene expression thereof, a CRISPR-associated DNA endonuclease with PAM interacting domain amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein, and an isolated, engineered Streptococcus pyogenes Cas9 (Spy Cas9) protein with a PID as either the PID amino acid composition of the isolated Streptococcus macacae Cas9 (Smac Cas9) protein or of a CRISPR-associated DNA endonuclease with PID amino acid sequences that are at least 80% identical to that of the isolated Streptococcus macacae Cas9 protein. A method for altering expression of at least one gene product employs Streptococcus macacae Cas9 endonucleases in complex with guide RNA, for specific recognition and activity on a DNA target immediately upstream of either an “NAA” or “NA” or “NAAN” PAM sequence.

Abstract: The invention provides compositions and methods for repeatable directed endonucleases (RDEs) and methods for repeatedly, and specifically cleaving DNA offset from the RDE's DNA recognition sequence on the target nucleic acid rather than within the DNA recognition sequence. Conservation of the recognition sequence of the target nucleic acid enables for re-localization of an RDE back to the DNA recognition sequence for further cleavage. The RDEs and methods of the invention are useful in applications including, but not limited to, recording data into a genome, timing the order of biochemical pathway events, efficient genome engineering and encoding lagged cellular death.

Abstract: Engineered Streptococcus canis Cas9 (ScCas9) variants include an ScCas9 protein with its PID being the PID amino acid composition of Streptococcus pyogenes Cas9 (SpCas9)-NG, an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 in its amino acid sequence (Sc+), and an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 and a substitution of residues ADKKLRKRSGKLATE in position 365-379 in the ScCas9 open reading frame (Sc++). Also included are CRISPR-associated DNA endonucleases with a PAM specificity of 5?-NG-3? or 5?-NNG-3? and a method of altering expression of a gene product by utilizing the engineered ScCas9 variants.

Abstract: A Streptococcus canis Cas9 (ScCas9) ortholog and its engineered variants, possessing novel PAM specificity, is an addition to the family of CRISPR-Cas9 systems. ScCas9 endonuclease is used in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence. A novel DNA-interacting loop domain within ScCas9, and other Cas9 orthologs, such as those from Streptococcus gordonii and Streptococcus angionosis facilitates a divergent PAM sequence from the “NGG” PAM of SpCas9.

Abstract: The invention provides compositions and methods for repeatable directed endonucleases (RDEs) and methods for repeatedly, and specifically cleaving DNA offset from the RDE's DNA recognition sequence on the target nucleic acid rather than within the DNA recognition sequence. Conservation of the recognition sequence of the target nucleic acid enables for re-localization of an RDE back to the DNA recognition sequence for further cleavage. The RDEs and methods of the invention are useful in applications including, but not limited to, recording data into a genome, timing the order of biochemical pathway events, efficient genome engineering and encoding lagged cellular death.